The present invention relates generally to the aging of proteins and other biomolecules resulting from reaction of glucose, and particularly to the non-enzymatic glycation or glycosylation of proteins and other susceptible amine-presenting molecules and subsequent reactions leading to advanced glycosylation end products, and to methods and agents for their inhibition.
The reaction between glucose and proteins has been known for many years. Its earliest manifestation was in the appearance of brown pigments during the cooking of food, which was identified by Maillard in 1912, who observed that glucose or other reducing sugars react with amino-containing compounds, including amino acids and peptides, to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments.
In the years that followed the initial discovery by Maillard, food chemists studied this reaction in detail and determined that stored and heat-treated foods undergo non-enzymatic browning as a result of the reaction between glucose and the polypeptide chain, and that the proteins are resultingly crosslinked and correspondingly exhibit decreased bioavailability. At this point, it was determined that the pigments responsible for the development of the brown color that develops as a result of protein glycosylation possessed characteristic spectra and fluorescent properties.
The reaction between reducing sugars and food constituents discussed above was found in recent years to have its parallel in vivo. Thus, the non-enzymatic reaction between aldehyde sugars such as glucose, galactose and arabinose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, is known as the Amadori product. In the case of ketone sugars such as fructose, this non-enzymatic reaction product is known as the Heyns product, with reactivities parallel to that of an Amadori product. This reaction has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminus of the .beta.-chain of hemoglobin, following an initial reaction with glucose, forms the modified hemoglobin known as hemoglobin A.sub.1c. Similar reactions have also been found to occur with a variety of other peptides, proteins, both soluble and structural, and biomolecules, such as lens crystallins, collagen nerve proteins, and low density lipoproteins, DNA and aminophopholipids.
As a result of the recent interest in this area, the first few stages of the Maillard reaction, and a relatively limited number of associated initial adducts and products, have become well-known. As subsequent reactions (including various dehydrations, oxidations, eliminations, condensations, cleavages, and other chemical changes) occur, however, a bewildering array of "early" and "late" glycation adducts and reactants is generated, and these are less well understood in molecular detail. As a group, the more advanced glycation adducts can be described as a class of yellow-brown, fluorescent pigments with intra- and intermolecular crosslinking activity, wherein specific glycation entities are thought to occur at low abundance within the widely divergent pool of advanced glycation end products (or AGEs). Despite significant work over the last twenty years or so, the molecular structures of only a few of these later glycation adducts and products have been determined, and the contribution of identified, in vivo-formed advanced glycation structures to specific biological processes remains poorly understood.
In U.S. Pat. No. 4,665,192 the fluorescent chromophore 2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole was isolated and identified from certain browned polypeptides such as bovine serum albumin and poly-L-lysine. This chromophore made possible the identification of the advanced glycosylation end products and assisted additional investigations seeking to clarify the protein aging process and to identify the specific chemistry involved in order to develop methods and agents for its inhibition.
More recently, other advanced glycation products have been identified, such as Farrnar et al., U.S. Pat. No. 5,017,696; pyrraline (Hayase et al., "Aging of Proteins: Immunological Detection of a Glucose-derived Pyrrole Formed during Maillard Reaction in Vivo", J. Biol. Chem., 263, pp. 3758-3764 (1989)); and pentosidine (Sell et al., "Structure Elucidation of a Senescence Cross-link from Human Extracellular Matrix", J. Biol. Chem., 264, pp. 21597-21602 (1989)).
A large body of evidence has been assembled to show that Maillard products as a whole underlie a wide variety of both normal and pathogenic activities and responses that occur as advanced glycation end products (or AGEs) accumulate in vivo. Such activity may be direct, as a consequence of the chemical reactivity of glycation products and adducts, or indirect, mediated by the cellular recognition of glycation adducts and products via AGE-specific binding proteins or receptors. An appreciation for the pathogenic potential of AGEs has suggested that interference with, or inhibition of, advanced glycation chemistry could be of enormous therapeutic benefit. The agent pimagedine (aminoguanidine), and other related compounds, have been found to be useful glycation inhibitors. This compound, and others like it, has been theorized to react with the carbonyl moiety of the early glycosylation product of a target protein formed subsequent to the initial non-enzymatic reaction with glucose or another reducing sugar, and thereby prevent further reaction to form open-carbonyl-containing advanced glycosylation end products.
Although pimagedine has shown a great therapeutic potential, there exist a need to discover and develop alternative glycation inhibitors, active, for instance, at different stages of the Maillard reaction and/or against a different spectrum of glycation intermediates and AGEs. Such alternates would provide additional treatment modalities against the deleterious sequelae of AGE accumulation in vitro and in vivo. The present invention is thus directed toward inhibition of the Maillard reaction, and is shown to operate through a mechanism not exploited previously in this regard.
Recently, it has been discovered that other naturally-occurring reducing sugars, including fructose, ribose and galactose, participate in non-enzymatic glycation and cross-linking. Because the methods and agents of the present invention block non-enzymatic crosslinking mediated by any such reactive sugars, they are expected to prevent fructose-mediated crosslinking as well. Cross-linking caused by other reactive sugars present in vivo or in foodstuffs, including ribose and galactose, would also be prevented by the methods and compositions of the present invention.